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The cAMP-dependent protein kinase

The cAMP-dependent protein kinase (cAPK)


Protein kinases are enzymes involved in many regulatable cellular processes. They covalently modulate other substrate proteins by phosphorylating serine, threonine or tyrosine. The required phosphate group is transfered from ATP to the corresponding hydroxy function of the substrate polypeptide. In the active center of the catalytic subunit the phosphoester chain of ATP (or corresponding derivatives) is complexed by two divalent metal ions, Magnesium in the native cAPK. So far no crystal structure of the catalytic subunit of cAPK has become available which contains both ATP and a protein substrate. The investigation of the reaction mechanism is now possible by the means of semiempirical AM1 quantum mechanics due to the recently published Magnesium parameters.

phospho transfer in cAPK

Influence of key residues on the reaction mechanism of the cAMP-dependent protein kinase

M. C. Hutter, V. Helms


The reaction mechanism of the catalytic phosphoryl transfer of cAMP-dependent protein kinase (cAPK) was investigated by semiempirical AM1 molecular orbital computations of an active site model system derived from the crystal structure of the catalytic subunit of the enzyme. The activation barrier is calculated as 20.7 kcal mol-1 and the reaction itself to be exothermic by 12.2 kcal mol-1. The active site residue Asp166 which was often proposed to act as a catalytic base does not accept a proton in any of the reaction steps. Instead, the hydroxyl hydrogen of serine is shifted to the simultaneously transferred phosphate group of ATP. Although the calculated transition state geometry indicates an associative phosphoryl transfer, no concentration of negative charge is found. To study the influence of protein mutations on the reaction mechanism, we compared two-dimensional energy hyper surfaces of the protein kinase wild-type model and a corresponding mutant in which Asp166 was replaced by alanine. Surprisingly, they show similar energy profiles despite the experimentally known decrease of catalytic activity for corresponding mutants. Furthermore, a model structure was examined, where the charged NH3 group of Lys168 was replaced by a neutral methyl group. The energetic hyper surface of this hypothetical mutant shows two possible pathways for phosphoryl transfer which both require significantly higher activation energies than the other systems investigated, while the energetic stabilization of the reaction product is similar in all systems. As the position of the amino acid side chains and the substrate peptide is virtually unchanged in all model systems, our results suggest that the exchange of Asp166 by other amino acid is less important to the phosphoryl transfer itself, but crucial to maintain the configuration of the active site in vivo. The positively charged side chain of Lys168, however, is necessary to stabilize the intermediate reaction states, particularly the side chain of the substrate peptide.


cAMP-dependent protein kinase; reaction mechanism; phosphoryl transfer; semiempirical molecular orbital calculation;

See also:

M. C. Hutter, V. Helms Mechanism of Phosphoryltransfer of cAMP-Dependent Protein Kinase Studied by Quantum-Chemical Calculations
Poster presented at the Third European Symposium of the Protein Society, Garmisch-Partenkirchen (D) 1999

under construction

M. Hutter December, 1st 1999

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